Reductively activated CPP–PROTAC nanocomplexes enhance target degradation via efficient cellular uptake

Abstract

We developed a nanoparticle based on a cell-penetrating peptide-PROTAC conjugate with a disulfide linker, MZ1-R9, and dextran sulfate, enhancing cellular uptake and BRD4 degradation. This delivery platform significantly improves PROTAC bioavailability and offers a promising strategy to overcome membrane permeability challenges for targeted protein degradation.

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The proteolysis-targeting chimeras (PROTACs) are a novel therapeutic that enables the selective degradation of disease-related proteins via the ubiquitin–proteasome system.^1–4 PROTACs induce proteasomal degradation of target proteins particularly in oncology. PROTAC comprises two ligands—one that binds to an E3 ubiquitin ligase and another that recognizes a protein of interest (POI)—connected by a linker. Despite their therapeutic potential, with over 40 PROTACs currently being evaluated in clinical trials, including the estrogen receptor–targeting ARV-471 (Phase 3; ClinicalTrials.gov Identifier: NCT05909397),⁵ the clinical translation of PROTACs remains hindered by intrinsic physicochemical limitations.

PROTACs commonly exhibit large molecular weights (typically > Mw 800) and low membrane permeability.⁶ Notably, even minimized PROTACs often exceed Mw 650,⁷ leading to suboptimal pharmacokinetics. For instance, MZ1—a well-characterized BRD4-targeting PROTAC⁸ shows PAMPA permeability approximately 190-fold lower than its POI ligand JQ1 and 290-fold lower than the E3 ligand VH032.⁹ To address these challenges, several chemical modification strategies have been explored to improve the drug-like properties of PROTACs.¹⁰ Although extensive structure–activity relationship (SAR) studies of each PROTAC candidate, such as replacing amides with esters, or introducing conformational constraints, are useful,¹¹ cell-penetrating peptides (CPPs) offer another versatile delivering platform for otherwise impermeable cargos, including proteins, nucleic acids, and nanoparticles into cells.¹² CPPs are short, typically cationic peptides (<30 amino acids) capable of translocating across the cell membrane, even at low concentrations.¹³ Inspired by these capabilities, CPPs have been extensively investigated for their potential in enhancing drug delivery platforms, including peptide–drug conjugates (PDCs) and antibody–drug conjugates (ADCs), aiming to improve pharmacokinetics, target selectivity, and intracellular accessibility.¹⁴ This approach is consistent with the broader strategy of targeted delivery systems designed to increase the pharmacological precision of therapeutic agents.¹⁵ For example, CPP-conjugated peptide-based PROTACs have been reported by conjugating a targeting peptide for cancer.^16–18

Nanoparticles are superior in controlling blood circulation, and therefore, high functionality has been developed using peptide-based nanoparticles as well as in the case of PROTAC.^19,20 Nanoparticle-based delivery systems are also expected to increase the versatility of CPP-PROTACs without altering their molecular structures. These systems possibly improve cell permeability and toxicity of CPP by decreasing the required compound dosage and enable targeted delivery through functionalization similar to recent studies which have successfully applied various nanocarriers, including liposomes, polymeric micelles, and inorganic particles, for delivering PROTACs.^21,22

In this study, we aimed to develop a delivery system that integrates CPP conjugation and nanoparticle formation to improve intracellular delivery and therapeutic efficacy of PROTACs. We designed MZ1-R9, a conjugate of the model PROTAC, MZ1 and the cationic nonaarginine (R9)²³ CPP, linked via a disulfide bond that is cleavable under reductive intracellular conditions. This conjugate was subsequently complexed with dextran sulfate (DS)—a biocompatible anionic polysaccharide—via electrostatic interactions to form nanoparticles.²⁴ The resulting nanocomplex enhances cellular uptake and intracellular release of the active PROTAC moiety, potentially overcoming key delivery challenges and enabling broader therapeutic applications of PROTAC technology. This nanocomplex enhances cellular uptake via endocytosis and releases the active PROTAC intracellularly upon disulfide bond cleavage by glutathione (GSH) (Fig. 1). This strategy addresses key delivery challenges of PROTACs by enhancing membrane permeability through nanoparticle formulation of the peptide-PROTAC conjugate.

Fig. 1 Schematic representation of the intracellular release mechanism of MZ1-R9/DS nanoparticles. Upon cellular entry, glutathione (GSH) reduces the disulfide bond, activating MZ1-R9 and releasing the active PROTAC, MZ1.

MZ1-R9 was synthesized via copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between MZ1 bearing a terminal azide on a disulfide linker and R9 modified at its N-terminus with an alkyne group. To evaluate cellular uptake behavior, a fluorescent analog, FAM-R9, was synthesized by substituting the MZ1 moiety with 5(6)-carboxyfluorescein, as shown in Scheme 1.

Scheme 1 Synthesis of (a) MZ1-R9 and (b) chemical structure of FAM-R9.

The size and zeta potential of the complexes were measured by DLS in 10 mM HEPES buffer (pH 7.3) to assess their physicochemical properties (Table S1). The charge ratio (N/S ratio) is defined as the molar ratio of protonatable nitrogen (N) groups in the peptide to sulfo (S) groups in DS. The fluorescence intensity of FAM-R9/DS complexes increased at N/S from 1, indicating formation of complexes was saturated at N/S of 1 (Fig. S6). FAM-R9/DS complexes were prepared at N/S ratios of 1, 2, 4, and 8, and their hydrodynamic size and surface charge were characterized by different techniques (DLS for N/S = 1 to 8 and NTA for N/S = 1, Table S1 and Fig. S7). All complexes exhibited hydrodynamic diameters below 200 nm, with PDI values consistently less than 0.4, indicating moderate size distributions typically considered acceptable for nanoparticle formulations. The particle size and PDI remained relatively constant across the tested N/S ratios, indicating stable nanoparticle formation over the examined range. In contrast, the zeta potential increased with increasing charge ratio, rising from +22.6 mV at N/S = 1 to +36.7 mV at N/S = 8. Furthermore, MZ1-R9/DS complexes exhibited small particle sizes below 100 nm, suggesting the formation of stable and compact nanoparticles that are favorable to efficient cellular internalization.

Intracellular fluorescence of FAM-R9/DS complexes was measured to assess cellular uptake in MCF-7 cells. Fluorescence intensity in MCF-7 cells was quantified by flow cytometry after 24 hours of treatment (Fig. 2 and Fig. S8). The FAM-R9/DS complex (N/S = 1) exhibited significantly enhanced cellular uptake, with fluorescence intensity approximately five times higher than that of free FAM-R9 (p < 0.001). These results suggest that nanoparticle formation enhances peptide stability and markedly promotes its intracellular uptake. Furthermore, the uptake of DS as a function of the N/S ratio was evaluated using fluorescently labeled DS (Fig. S9). To ensure that the physicochemical properties of the complex matched those of FAM-R9/DS, R9 was labeled with 5-nitrofluorescein (5-NO₂-FAM)—a structural analog of FAM that lacks fluorescence—yielding 5-NO₂-FAM-R9. The highest cellular uptake was observed at an N/S = 1, mirroring the uptake profile of the peptide. These findings indicate that DS, along with the peptide, is internalized as nanoparticulate complexes. Taken together, these results highlight the importance of N/S ratio optimization in the design of nanocarriers for efficient intracellular delivery. Based on these findings, subsequent experiments were conducted using N/S ratios of 1 and 8.

Fig. 2 Intracellular uptake of FAM-R9 and FAM-R9/DS complexes in MCF-7 cells. The peptide concentration was fixed at 2 μM across all conditions, with varying volumes of 8 μM DS solution corresponding to N/S ratios of 1 (30 μL), 2 (15 μL), 4 (7.5 μL), and 8 (3.8 μL). Fluorescence intensity was measured by flow cytometry following incubation with free FAM-R9 or FAM-R9/DS complexes at different N/S ratios (n = 3, mean ± SD, ***p < 0.001).

Time-dependent intracellular uptake of FAM-R9 and FAM-R9/DS complexes was evaluated using flow cytometry (Fig. S10). The FAM-R9/DS complex (N/S = 1) exhibited markedly higher intracellular uptake than both free FAM-R9 and the complex at N/S = 8, with maximal accumulation observed at 12 hours post-treatment. This complex consistently maintained the highest intracellular uptake even after 24 hours, indicating superior and sustained cellular internalization at N/S = 1.

To elucidate the uptake mechanism, MCF-7 cells were treated with three endocytosis inhibitors: amiloride (5 mM), methyl-β-cyclodextrin (MβCD, 10 mM), and sucrose (0.4 M). Each inhibitor selectively blocks a distinct endocytic pathway: amiloride for macropinocytosis, MβCD for caveolae-mediated endocytosis, and sucrose for clathrin-mediated endocytosis.^25,26 MCF-7 cells were pretreated with each inhibitor at 37 °C for 30 minutes, followed by incubation with FAM-R9 or the FAM-R9/DS complex (N/S = 1) for an additional 30 minutes. Intracellular fluorescence was then quantified. The uptake of both FAM-R9 and its DS complex was significantly reduced by all three inhibitors, indicating that their cellular entry occurs predominantly via endocytosis (Fig. 3). Intracellular localization was examined by fluorescence microscopy. After 2 hours of treatment with the FAM-R9/DS complex (N/S = 1), minimal overlap between FAM-R9/DS signals and LysoTracker Red was observed, suggesting that punctate fluorescence pattern implies that the complex may remain in particulate form, which could potentially hinder the efficient cytosolic release of MZ1 (Fig. S11). Nonetheless, a fraction of the complex may be released from endosomes or lysosomes.

Fig. 3 Mechanistic analysis of cellular uptake of FAM-R9 and the FAM-R9/DS complex (N/S = 1) in MCF-7 cells using endocytosis inhibitors: amiloride (5 mM, macropinocytosis inhibitor), methyl-β-cyclodextrin (MβCD, 10 mM, caveolae-mediated endocytosis inhibitor), and sucrose (0.4 M, clathrin-mediated endocytosis inhibitor). Data are expressed as mean ± SD (n = 3), ***p < 0.001.

The in vitro stability of MZ1-R9 under physiologically relevant conditions was evaluated. MZ1-R9 and the MZ1-R9/DS complex (N/S = 1) were dissolved in PBS containing 5 mM GSH at a final concentration of 100 nM and incubated at 37 °C. Aliquots were collected at different time points for UHPLC analysis. As shown in the drug release profiles (Table 1), free MZ1-R9 exhibited a rapid burst release of MZ1 within the first 30 minutes under reductive conditions, with nearly complete conversion to free MZ1. In contrast, the MZ1-R9/DS complex (N/S = 1) showed a sustained release profile, retaining approximately 13% of the intact conjugate after 6 hours. In addition, MZ1-R9/DS was more stable in serum-containing medium than MZ1-R9 (Fig. S13 and Table S2). These results indicate that the nanocomplex exhibits enhanced in vitro stability and enables gradual PROTAC release, supporting its potential for sustained intracellular delivery.

Table 1 UHPLC analysis of MZ1-R9 and the MZ1-R9/DS complex (N/S = 1) after incubation in PBS containing 5 mM GSH at 37 °C. The peak areas of MZ1-R9 and released MZ1 were normalized to 100%

	Time (h)	MZ1-R9 (%)	MZ1 (%)
MZ1-R9	0	100	0
	0.5	0	100
	1	0	100
	2	0	100
	6	0	100
	24	0	100
MZ1-R9/DS (N/S = 1)	0	100	0
	0.5	32.9	67.1
	1	16.9	83.1
	2	30.6	69.4
	6	12.7	87.3
	24	0	100

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We next evaluated the BRD4 degradation activity of MZ1-R9 and its DS complexes using western blot analysis in MCF-7 cells. Cells were treated with the compounds, and BRD4 expression levels were subsequently assessed. To allow sufficient time for intracellular uptake, endosomal escape, disulfide bond reduction, and subsequent release and target degradation, the experimental protocol was modified accordingly. Cells were treated with the compounds, followed by PBS washing to remove extracellular components. Following PBS washes at each time point, cells were further incubated for 24 hours to allow sufficient time for complete BRD4 degradation (Fig. 4a).

Fig. 4 (a) Experimental workflow for western blot analysis of BRD4 degradation by MZ1-R9 and its DS complexes (N/S = 1 and N/S = 8). Protein expression levels were quantified using anti-BRD4 and anti-β-actin antibodies. Numbers below the BRD4 bands indicate the BRD4/β-actin ratio, normalized to the vehicle control (set to 100%). MCF-7 cells were treated for (b) 6 h, (c) 12 h, and (d) 24 h.

Time-course analysis revealed that neither MZ1-R9 nor the MZ1-R9/DS complex (N/S = 1) induced detectable BRD4 degradation after 6 hours of treatment. However, after 12 hours, the N/S = 1 complex exhibited BRD4 degradation comparable to that of unconjugated MZ1. Furthermore, at both 12 and 24 hours, the N/S = 1 complex induced greater BRD4 degradation than MZ1-R9 alone, suggesting that nanoparticle formation facilitated improved intracellular delivery and enhanced degradation efficiency (Fig. 4b–d).

To further assess intracellular delivery efficiency, we performed LC-MS/MS analysis. MCF-7 cells were treated with 100 nM of either free MZ1 or the MZ1-R9/DS complex (N/S = 1) for 12 hours, followed by 24-hour incubation to ensure complete uptake and intracellular processing. LC-MS/MS quantification revealed that nanoparticle formation enhanced intracellular MZ1 levels by approximately 4-fold compared to free MZ1. Specifically, free MZ1 achieved a cellular uptake of 0.049% of the administered dose, whereas the MZ1-R9/DS complex (N/S = 1) reached 0.21%, confirming improved cellular accumulation (Fig. 5).

Fig. 5 LC-MS/MS quantification of intracellular MZ1 levels in MCF-7 cells following 12-hour treatment with MZ1, or the MZ1-R9/DS complex (N/S = 1). Data are presented as mean ± SD (n = 3), **p < 0.01.

Our CPP-conjugated PROTAC nanoparticles enhance intracellular delivery and target protein degradation through a dual mechanism: membrane translocation mediated by CPP and endocytosis facilitated by nanoparticle formation with DS. This strategy aligns with previous studies demonstrating the efficacy of CPPs in facilitating PROTAC uptake, including Tat-linked FOXM1 degraders,²⁷ polyarginine-tagged CREPT degraders,²⁸ and CPP-fused ERα degraders.^16,17 However, unlike these covalently conjugated systems, our approach leverages noncovalent electrostatic interactions with DS, enabling flexible and modular nanoparticle formation that can be adapted to a variety of PROTAC structures without complex synthetic procedures. Because the conjugation is achieved via noncovalent interactions with DS, it allows modular adaptation to various PROTACs without requiring synthetic modification. Compared to other advanced nanodelivery systems, such as POLY-PROTACs²⁹ or LNP-encapsulated bioPROTACs,³⁰ our DS-based platform offers a simpler and more accessible route for formulation. Unlike LNPs, which require complex formulation and often pose stability issues, or covalent polymer–based systems that demand chemical modification of the PROTAC itself, our noncovalent DS-based approach enables rapid assembly, avoids chemical derivatization, and retains compatibility with diverse PROTAC structures. The resulting nanoparticles possess favorable physicochemical properties, including a hydrodynamic diameter of approximately 90 nm at an N/S ratio of 1 and a moderately positive surface charge, both of which promote efficient cellular uptake via multiple endocytic pathways—consistent with previous reports on polyplex-based delivery systems. Despite a marked ∼4-fold increase in intracellular MZ1 concentration achieved with our nanoparticle system, the corresponding enhancement in BRD4 degradation was not proportional. This discrepancy suggests that additional intracellular barriers—such as incomplete endosomal escape, limited nuclear translocation, or suboptimal disulfide cleavage—may hinder effective target engagement and degradation. Several lines of evidence from recent literature support these possible bottlenecks. First, numerous studies have demonstrated that CPP-conjugated macromolecules are often entrapped within endosomes following internalization, resulting in inefficient cytosolic and nuclear delivery.^31,32 Our DS-based nanoparticle system, although effective in facilitating uptake, lacks a dedicated endosomolytic component, which could limit the release of MZ1 into the cytosol and thereby reduce its access to both BRD4 and E3 ligases. In addition, the disulfide linker used for redox-responsive release may cleave too slowly under physiological conditions.^33,34 Our UHPLC data show that approximately 13% of the MZ1-R9 conjugate remained intact after 6 hours in reductive buffer, suggesting that gradual release may limit the availability of active MZ1. Similar release kinetics have been identified as limiting factors in related PROTAC delivery systems.³⁵ Taken together, these factors may explain why increased cellular MZ1 concentration does not translate linearly to enhanced BRD4 degradation. To address these bottlenecks, future modifications could incorporate endosomolytic motifs, nuclear localization sequences, or dynamic linkers optimized for rapid and efficient intracellular release of the active PROTAC.

In summary, we have developed a modular and redox-responsive nanocomplex platform that significantly improves intracellular delivery and modestly enhances target degradation efficiency. By conjugating the model PROTAC, MZ1, to R9 and assembling the resulting conjugate into nanoparticles with DS, we achieved enhanced intracellular uptake, sustained release, and improved BRD4 degradation in vitro. This delivery strategy requires no covalent modification to the carrier polymer, offering versatility and ease of adaptation to other PROTAC structures.

PolyPROTACs and nanocarriers covalently linked to PROTACs achieve controlled release through chemical modification strategies. On the other hand, our modular, electrostatically assembled design, while potentially sensitive to physiological ionic strength and serum conditions, enables the flexible incorporation of diverse functional peptides into a single structure, thereby avoiding complex molecular modifications and enhancing the potential of multifunctional delivery systems. Additionally, the redox-sensitive properties enable the release of cargo under reduced intracellular conditions.

While this platform significantly increases intracellular PROTAC levels, the enhancement in degradation activity was not strictly proportional, indicating that further optimization—such as endosomal escape enhancers or cleavable linkers tailored for intracellular release—may be necessary. Looking forward, integration of targeting ligands, responsive linkers, or combinatorial payloads may further refine the selectivity and efficacy of this system. Our findings establish a foundational delivery strategy that addresses a critical bottleneck in PROTAC development and opens new avenues for targeting intracellular disease drivers. To address current limitations in intracellular trafficking and degradation, future studies will focus on optimizing cleavable linkers, incorporating endosomal escape enhancers, and evaluating critical factors such as GSH-dependent cleavage efficiency and the potential requirement for nuclear localization in BRD4 degradation.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5cb00196j

Acknowledgements

This study was supported in part by AMED under grant numbers 25mk0121286 and 25ama221127 to Y. D. and JP25am0521016 and JP25mk0121325 to H. Y. This work was also supported by the Japan Society for the Promotion of Science (KAKENHI, grants JP21K05320 and JP23H04926 to Y. D. and JP24K18260 to H. Y.), JST (ACT-X, grant number JPMJAX222L to H. Y.), Takeda Science Foundation to H. Y and Astellas Foundation for Research on Metabolic Disorders to H. Y.

Notes and references

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